Methods for the production of hybrid seed

ABSTRACT

The present invention provides a dual method for producing male-sterile plants. Two genetically transformed plants, parents 1 and 2 are crossed to obtain male-sterile offspring. Parent 1 is transformed with an expression cassette comprising a nucleotide sequence encoding an anther-specific promoter which is operably linked to a nucleotide sequence encoding a transactivator. Parent 2 is transformed with an expression cassette comprising a target nucleotide sequence, which is capable of being activated by the transactivator, operably linked to a nucleotide sequence which encodes RNA or a polypeptide which will disrupt the formation of viable pollen. Therefore, crossing parent 1 with parent 2 results in male-sterile offspring. The male-sterile plants are useful for producing hybrid seed. 
     The invention also provides compositions and methods for high level expression of a coding region of interest in a plant.

This is a divisional application of Ser. No. 08/368,773, filed Jan. 3,1995, now U.S. Pat. No. 5,659,124 which is a divisional of Ser. No.07/950,348, filed Sep. 24, 1992, now U.S. Pat. No. 5,409,823.

FIELD OF THE INVENTION

The invention relates to the production of male-sterile plants and theuse of such plants in producing hybrid seed.

BACKGROUND OF THE INVENTION

Heterosis in corn has received considerable attention because of itsmarked effect on yield improvement. This increased productivity oncrossing different strains of corn was first noted in the late 19thcentury and was then developed according to systematic geneticprocedures.

The usual method for raising hybrid corn is to establish many inbredlines, make intercrosses, and determine which hybrids are moreproductive in a given locality.

The success of hybrid maize motivated plant breeders to explore theexistence and magnitude of hybrid vigor in many other species witheconomic importance. In general, hybrids increase yields. They areusually more efficient in use of growth factors and give a greaterreturn per unit for the growth factors such as water and fertilizer.Under stress F₁ hybrids are generally superior to parental cultivars,with a more stable performance over a wide range of environments. Withhybrids, there is uniformity in product and maturity that oftenfacilitates harvest and increases the value of the product in themarketplace. The F₁ hybrid may combine characters that are difficult orimpossible to combine in other ways. This is particularly true of manyinterspecific and intergeneric hybrids. The general conclusion fromresearch is that hybrid vigor, a common phenomenon in plants is ofsufficient magnitude to warrant commercial exploitation if appropriatetechniques can be devised.

Hybrid vigor has been recognized as a wide-spread phenomenon in plantsand animals for many years. Commercial hybrids are now used extensivelyin many crops, including corn, sorghum, sugarbeet, and sunflower.Research is being conducted on many other crops that may permit thewide-spread use of commercial hybrids in the future.

Commercial hybrids have the greatest potential for crops in which thehybrid seed can be produced reliably and economically. Three biologicalrequirements for successful hybrid seed production include the presenceof hybrid vigor, elimination of fertile pollen in the female parent, andadequate pollination by the male parent.

In order to produce hybrid seed uncontaminated with selfed seed,pollination control methods must be implemented to ensurecross-pollination and not self-pollination. Known pollination controlmechanisms are generally mechanical, chemical, or genetic.

Elimination of fertile pollen from the female parent can be achieved byhand emasculation in some species such as maize, a monoecious species.Such elimination of fertile pollen is achieved by pulling or cutting themale inflorescence (tassel) from plants in the female parent population.This simple procedure prevents self-fertilization by mechanicallydetasseling female plants before pollen shed to prevent selfing.However, most major crop plants of interest have both functional maleand female organs within the same flower. Thus, emasculation is not asimple procedure. At any rate, this form of hybrid seed production isextremely labor intensive and hence expensive.

To eliminate the laborious detasseling that is necessary to preventself-fertilization in hybrid crosses, cytoplasmic factors which producemale-sterility have been used in some species in conjunction withrestorer genes.

Male-sterility in the female parent can be controlled by nuclear genesor by a cytoplasmic-genetic system. Genetic male-sterility is controlledby nuclear genes in which the alleles for sterility generally arerecessive to the alleles for fertility. Genetic male-sterility occurs inmany species. Usually, it is controlled by a single recessive gene thatmust be homozygous to cause male-sterility. Breeders who use geneticmale-sterility for hybrid seed production usually develop aphenotypically uniform female line that segregates 1:1 for Msms and noMsms individuals. Seed for these lines is increased in isolation byharvesting seed from msms plants that are pollinated from Msms plants.To produce commercial F₁ hybrid seed with genetic male-sterility, the 50percent of female Msms plants must be rouged from the field as soon astheir fertility can be identified. The labor associated with rougingfertile plants from female plants has greatly restricted the use ofgenetic male-sterility in producing hybrid seed. There are severalproblems associated with this system for producing commercial hybridseed. First, it is not possible to eliminate fertile plants from thedesired male-sterile plants in the female population. Geneticmale-sterile plants must be maintained by mating them with male-fertileindividuals. Half of the F₁ plants from such a cross would be sterile,but the remaining plants would be fertile. Thus, the unwantedmale-fertile plants in the female population may disseminate pollen andreduce the effectiveness of the desired male parent.

The successful use of cytoplasmic male-sterility for commercial hybridseed production requires a stable male-sterile cytoplasm, an adequatepollen source, and an effective system of getting the pollen from themale parent to the male-sterile female. Also, the cytoplasmic-geneticsystem of male sterility requires three lines to produce a singlecrossed hybrid; the A line (male-sterile), B line (male-fertilemaintainer), and R line (male-fertile with restorer genes). Three-waycrosses produced with cytoplasmic-genetic male sterility involvedmaintenance and production of four lines, an A and B line of one inbredand male-fertile inbreds of the other two.

Furthermore, the southern corn blight caused by Helminthosporium maydis,Race T, which severely attacked all maize hybrids with cytoplasmicmale-sterile T cytoplasm, demonstrated the vulnerability of a hybridseed production industry based on a single source of male-sterilecytoplasm. For hybrid maize, most seed producers have returned to handor mechanical emasculation and wind pollination.

Hybrid seed may also be produced by the use of chemicals that block orkill viable pollen formation. These chemicals, gametocides, are used toimpart a transitory male-sterility. However, the expense andavailability of the chemicals and the reliability of the applicationslimits the production of hybrid seed by using gametocides.

Molecular methods for hybrid seed production have also been described.Such methods transform plants with constructs containing anti-sense DNAand other genes which are capable of controlling the production offertile pollen into plants. Such regenerated plants are functionallymale-sterile and are used for the production of hybrid seed by crossingwith pollen from male-fertile plants. The primary deficiencies of theseapproaches stem from the fact that the genetically engineered malesterility gene, whether it is an anti-sense or RNAse, can only bemaintained in a heterozygous state. They are fundamentally the same asnatural genetic male steriles in that they must be maintained bycrossing to isogenic male fertile lines. This is most problematic in thehybrid cross field where the acreage is large and yield is critical. Theheterozygous female parent, of which only 50% will be male sterile, mustbe planted in rows next to the pollen donor male parent and the 50%fertile female parents removed. This is rendered easier in geneticallyengineered genetic male steriles because a herbicide resistance gene canbe linked to the male sterility gene, and herbicide spray can be used toremove the fertile plants, but it still means that the female parentrows must be planted at double density in order to get the same yieldper acre of our system. This will cause some yield loss due tocompetition. The herbicide spray also means yield loss because theresistant plants are never 100% immune to the herbicide, and the costsof spraying the chemical are considerable.

Accordingly, there is a need for a reliable simple technique for theformation of hybrid seed production.

SUMMARY OF THE INVENTION

The present invention is drawn to a method for producing male-sterileplants. The method comprises crossing two genetically transformedplants, parents 1 and 2. Parent 1 is transformed with an expressioncassette which comprises a nucleotide sequence which encodes a firstpolypeptide, a transactivator, capable of regulating a second nucleotidesequence, a target nucleotide sequence. The DNA sequence encoding thefirst polypeptide is operably linked to an anther specific promoter.

Parent 2 is transformed with an expression cassette which comprises thetarget nucleotide sequence operably linked to a nucleotide sequencewhich encodes RNA or a polypeptide, both of which are capable ofdisrupting the formation of viable pollen. When parents 1 and 2 arecrossed, polypeptide 1, the transactivator, regulates the targetnucleotide sequence and turns on the expression of polypeptide 2. Thus,no viable pollen is formed in the subsequent generation.

The male-sterile plants are useful for producing hybrid seed.

The invention is further drawn to compositions and methods for highlevel expression of a heterologous gene in plants. In this manner, afirst construct comprises a 5' regulatory region of interest operablylinked to a nucleotide sequence which encodes a T7 polymerase. A secondconstruct comprises the coding region of a polypeptide of interestoperably linked to a T7 5' regulatory region. When a plant has beentransformed with both constructs, high level expression of thepolypeptide of interest is regulated by the T7 polymerase. By utilizingspecific plant promoters to direct expression of the T7 polymerase, highlevels of a polypeptide or RNA of interest can be obtained in specifictissues or at specific developmental stages.

DETAILED DESCRIPTION OF THE INVENTION

A dual system for production of male-sterile plants is provided. Thesystem involves crossing two genetically transformed plants, hereinreferred to as parents 1 and 2. Parent 1 is transformed with anexpression cassette which comprises a nucleotide sequence which directsthe expression of a first polypeptide in anthers. This first polypeptideis capable of regulating the transcription of a second nucleotidesequence, the target DNA, which directs expression of RNA or a secondpolypeptide capable of disrupting the production of viable pollen.Parent 2 is transformed with an expression cassette which comprises thetarget nucleotide sequence operably linked to a nucleotide sequencewhich encodes RNA or a polypeptide capable of disrupting the formationof viable pollen.

As noted, the RNA or the second polypeptide can only be expressed whenin the presence of the first polypeptide, the transactivator. Thus, bothparents 1 and 2 are male-fertile. However, upon crossing parent 1 withparent 2, the transactivator regulates the expression of the RNA orpolypeptide 2 via the target DNA sequence. The result is no viablepollen is produced. The resulting progeny containing both expressioncassettes are male sterile.

Male sterility is the failure or inability to produce functional orviable pollen. Male sterility may result from defects leading to thenon-formation of pollen or to the lack of functional ability in thepollen when it is formed. Therefore, either pollen is not formed or, ifformed, it is either non-viable or incapable of effective fertilizationunder normal conditions.

The male-sterile plants of the invention, are female fertile. That is,the plants do not produce fertile pollen, yet are capable of acceptingpollen from the desired paternal parent resulting in fertilization andseed production.

There are several transactivator polypeptides which can be used in thepresent dual sterility system. The important aspect is that the RNA orthe second polypeptide which disrupts pollen formation is not expressedin the absence of the first or transactivator polypeptide.

The transactivators of the invention are capable of activating a targetnucleotide sequence which is operably linked to a nucleotide sequencewhich encodes RNA or a second polypeptide both of which are capable ofdisrupting the production of viable pollen. Thus, the nucleotidesequence operably linked to the target sequence is only expressed in thepresence of the transactivator.

The transactivators of the invention include, but are not limited to,polymerases, DNA binding proteins, naturally occurring and synthetictranscriptional activators, translational activators,post-transcriptional activators, and the like. The use of suchtransactivator polypeptides in directing expression of anothernucleotide sequence is exemplified by the T7 RNA polymerase. See, U.S.Pat. Nos. 5,122,457; 5,126,251; and 5,135,855; Lassner et al., (1991)Plant Molecular Biology 17:229-234; Rodriguez et al., (1990) Journal ofVirology 64:4851-4857; Vennema et al., (1991) Gene 108:201-210; Bentonet al., Molecular and Cellular Biology (1990) Molecular and CellularBiology 10:353-360; Elroy-Stein and Moss (1990) Proceedings, Proc. Natl.Acad. Sci.:USA 87:6743-6747; Moss et al., (1990) Nature 348:91-92;Elroy-Stein et al., (1989) Proceedings, Proc. Natl. Acad. Sci.:USA86:6126-6130; and Rosenberg et al., (1987) Gene 56:125-135, hereinincorporated by reference.

Regulator polypeptides or transactivators also include DNA bindingproteins which are necessary for transcription activation of specificpromoters. Binding domains of one protein may be fused to activitydomains of another protein to form chimeras of such DNA bindingproteins, such as GAL4/VP16 (Carey et al. (1989), J. Mol. Biol.,209:423-432; Cress et al. (1991) Science, 251:87-90; and Sadowski et al.(1988), Nature, 335:563-564). Likewise, the binding domain of otherproteins, i.e., Lex A (Brent and Ptashne, (1985), Cell, 43:729-736,which describes a Lex A/GAL4 transcriptional activator) can be utilized.

Translational activators are exemplified by the cauliflower mosaic virustranslational activator (TAV). See, for example Futterer and Hohn (1991)EMBO J. 10:3887-3896. In this system a dicistronic mRNA is produced.That is, two coding regions are transcribed in the same mRNA from thesame promoter. In the absence of TAV, only the first cistron istranslated by the ribosomes. However, in cells expressing TAV, bothcistrons are translated. The coding region for a polypeptide capable ofdisrupting the formation of viable pollen is placed in the position ofthe second cistron.

The expression cassette, expression cassette 1, utilized to transformparent 1 comprises an anther 5' regulatory region operably linked to thefirst polypeptide, the transactivator. The 5' regulatory regions of theinvention include nucleotide sequences necessary for expression, i.e.the promoter region. The construct may also include any other necessaryregulators such as terminators, (Guerineau et al., (1991), Mol. Gen.Genet., 226:141-144; Proudfoot, (1991), Cell, 64:671-674; Sanfacon etal., (1991), Genes Dev., 5:141-149; Mogen et al., (1990), Plant Cell,2:1261-1272; Munroe et al., (1990), Gene, 91:151-158; Ballas et al.,(1989), Nucleic Acids Res., 17:7891-7903; Joshi et al., (1987), NucleicAcid Res., 15:9627-9639); nuclear localization signals (Kalderon et al.,(1984) Cell, 39:499-509; and Lassner et al., (1991) Plant MolecularBiology, 17:229-234); plant translational consensus sequences (Joshi, C.P., (1987), Nucleic Acids Research, 15:6643-6653), introns (Luehrsen andWalbot, (1991), Mol. Gen. Genet., 225:81-93) and the like, operablylinked to the nucleotide sequence of the transactivator.

The expression cassette, expression cassette 2, utilized to transformparent 2 comprises a nucleotide sequence upon which the transactivatoracts operably linked to a coding region of interest. Additionalregulating nucleotide regions may also be included, such as terminators,promoters, leader sequences and the like. Such regions are operablylinked to the coding region.

It may be beneficial to include 5' leader sequences in the expressioncassette 2 construct. Such leader sequences can act to enhancetranslation. Translational leaders are known in the art and include:

Picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5'noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. (1989)PNAS USA 86:6126-6130);

Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allisonet al., (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology,154:9-20), and

Human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D. G.,and Sarnow, P., (1991), Nature, 353:90-94;

untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4), (Jobling, S. A., and Gehrke, L., (1987), Nature,325:622-625;

Tobacco mosaic virus leader (TMV), (Gallie, D. R. et al., (1989),Molecular Biology of RNA, pages 237-256; and

Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al.,(1991), Virology, 81:382-385. See also, Della-Cioppa et al., (1987),Plant Physiology, 84:965-968.

Either a plant terminator, a T7 terminator or both may be utilized inexpression cassette 2. See, Rosenberg et al., (1987), Gene, 56:125;Guerineau et al., (1991), Mol. Gen. Genet., 226:141-144; Proudfoot,(1991), Cell, 64:671-674; Sanfacon et al., (1991), Genes Dev.,5:141-149; Mogen et al., (1990), Plant Cell, 2:1261-1272; Munroe et al.,(1990), Gene, 91:151-158; Ballas et al., (1989), Nucleic Acids Res.,17:7891-7903; Joshi et al., (1987), Nucleic Acid Res., 15:9627-9639.

Particular expression cassettes will be discussed in more detail fordifferent transactivator systems and exemplified in the experimentalsection.

In one embodiment of the invention, a T7 polymerase system is utilized.The T7 bacteriophage harbours a gene coding for an RNA polymerase whichrecognizes a phage specific promoter. The polymerase and the phagepromoters have unique properties which prevent interference withexpression of host genes.

The T7 RNA polymerase is a monomeric enzyme of 100 kD whereas most otherpolymerases are more complex. The T7 promoter consists of 23 bp whichare not encountered in other prokaryotic or eukaryotic promoters. See,Dunn et al., (1983) J. Mol. Biol. 166:477-535; Davanloo et al., (1984)Proceedings Proc. Natl. Acad. Sci.:USA 81:2035-2079; and Moffatt et al.,(1984) J. Mol. Biol. 173:265-269.

To use a T7 polymerase system to produce male-sterile plants, parent 1is transformed with an expression cassette comprising an anther 5'regulatory region operably linked to a nucleotide sequence encoding theT7 polymerase. A nuclear location signal (NLS) such as the SV40 nuclearlocation signal may also be incorporated into the construct. See, forexample, Kalderon et al., (1984) Cell 39:499-509; Dunn et al., (1988)Gene 68:259-266; Hunt T. (1989) Cell 59:949-951; and Lassner et al.,(1991) Plant Molecular Biology 17:229-234, which disclosures are hereinincorporated by reference. A plant translational consensus sequence(Joshi, C. P. (1987) Nucleic Acids Research 15:6643-6653) may be alsoincluded, as well as plant termination signals and introns.

Anther-specific promoters are known in the art. By utilizinganther-specific promoters, the resulting transgenic plants will expressthe T7 polymerase only in the anther of the plant. Anther-specificpromoters are set forth in U.S. application Ser. No. 908,242 filed Jul.2, 1992, which disclosure is herein incorporated by reference.

In the case of promoter DNA sequences, "anther-specific" describesregulatory sequences which direct the transcription of associated codingsequences so that the corresponding messenger RNA is present in anthertissue in concentrations at least about 100-fold that observed in othertissues.

The expression cassette utilized to transform parent 2 comprises a T7promoter operably linked to a nucleotide sequence which encodes RNA or apolypeptide which disrupts formation of viable pollen when expressed.Such polypeptides include but are not limited to:

Diphtheria Toxin A-chain (DTA), which inhibits protein synthesis,Greenfield et al., (1983), Proc. Natl. Acad., Sci.:USA, 80:6853;Palmiter et al., (1987), Cell, 50:435;

Pectate lyase pelE from Erwinia chrysanthemi EC16, which degradespectin, causing cell lysis. Keen et al., (1986), J. Bacteriology,168:595;

T-urf13 (TURF-13) from cms-T maize mitochondrial genomes; this geneencodes a polypeptide designated URF13 which disrupts mitochondrial orplasma membranes. Braun et al., (1990), Plant Cell, 2:153; Dewey et al.,(1987), Proc. Natl. Acad. Sci.:USA, 84:5374; Dewey et al., (1986), Cell,44:439;

Gin recombinase from phage Mu a gene, which encodes a site-specific DNArecombinase which will cause genome rearrangements and loss of cellviability when expressed in cells of plants. Maeser et al., (1991), Mol.Gen. Genet., 230:170-176;

Indole acetic acid-lysine synthetase (iaaL) from Pseudomonas syringae,which encodes an enzyme that conjugates lysine to indoleacetic acid(IAA). When expressed in the cells of plants, it causes altereddevelopments due to the removal of IAA from the cell via conjugation.Romano et al., (1991), Genes and Development, 5:438-446; Spena et al.,Mol. Gen. Genet., (1991), 227:205-212; Roberto et al., Proc. Natl. Acad.Sci.:USA, 87:5795-5801; and,

CytA toxin gene from Bacillus thuringiensis Israeliensis which encodes aprotein that is mosquitocidal and hemolytic. When expressed in plantcells, it causes death of the cell due to disruption of the cellmembrane. McLean et al., (1987), J. Bacteriology, 169:1017-1023; Ellaret al., (1990), U.S. Pat. No. 4,918,006.

Such polypeptides also include Adenine Phosphoribosyltransferase (APRT)(Moffatt and Somerville, (1988), Plant Physiol., 86:1150-1154); DNAse,RNAse; protease; salicylate hydroxylase; etc.

It is further recognized that the T7 promoter could be operably linkedto RNA which is capable of disrupting the formation of viable pollen.The RNA of the invention includes antisense RNA as well as ribozymes.Antisense RNA can be utilized which will hybridize with mRNA from a genewhich is critical to pollen formation or function, i.e. APRT. In thismanner, the anti-sense RNA will prevent expression of the necessarygenes resulting in no pollen formation.

Alternately, ribozymes can be utilized which target mRNA from a genewhich is critical to pollen formation or function. Such ribozymes willcomprise a hybridizing region of at least about nine nucleotides whichis complementary in nucleotide sequence to at least part of the targetRNA and a catalytic region which is adapted to cleave the target RNA.Ribozymes are described in EPA No. 0 321 201 and WO88/04300 hereinincorporated by reference. See, also Haseloff and Gerlach, (1988),Nature, 334:585-591; Fedor and Uhlenbeck, (1990), Proc. Natl. Acad.Sci.: USA, 87:1668-1672; Cech and Bass, (1986), Ann. Rev. Biochem.,55:599-629; Cech, T. R., (1987), 236:1532-1539; Cech, T. R. (1988) Gene,73:259-271; and, Zang and Cech, (1986), Science, 231:470-475.

When parent 1 is crossed with parent 2, the progeny contain bothexpression cassettes. Therefore, the T7 polymerase drives expression ofthe coding sequence under the control of the T7 promoter. A polypeptideor RNA is expressed which disrupts the formation of viable pollenresulting in male-sterility.

Having described the T7 polymerase system in detail, one of skill in theart will recognize that other transactivators may be utilized to obtainthe same effect.

Other known transactivators include, but are not limited to GAL4 (Careyet al., (1989), J. Mol. Biol., 209:423-432; Ginger et al., (1985), Cell,40:767-774); VP16 (Cress and Triezenberg (1991), Science, 251:87-90);GAL4-VP16 (Sadowski et al., (1988), Nature, 335:563-564); etc. See also,Ma and Ptashne, (1987), Cell, 43:729-736; Hope and Struhl, (1986), Cell,46:885-894; and Gill and Ptashne, (1987), Cell, 51:121-126. Suchtransactivators activate transcription in yeast, plant, insect andmammalian cells. These proteins typically contain two parts. One partdirects DNA binding and the other, the activating region, presumablyinteracts with some component of the transcriptional machinery. Thus,fusions such as GAL4-VP16; GAL4-c1 may be utilized.

The transactivators GAL4/VP16 and GAL4/c1 can be utilized totransactivate a promoter with at least one GAL4 binding site. In thissystem, parent 1 is transformed with an expression cassette comprisingan anther 5' regulatory region operably linked to GAL4/VP16 or GAL4/c1.Parent 2 is transformed with an expression cassette comprising in 5' to3' orientation, a GAL4 binding site, a minimal promoter or 5' regulatoryregion and the coding region of interest. By minimal promoter isintended that the basal promoter elements are inactive or nearly sowithout upstream activation.

The offspring from a cross of parent 1 and parent 2 will be male-sterileas the GAL4 transactivator will direct expression of the polypeptide oranti-sense RNA which will disrupt formation of viable pollen.

As discussed earlier, translational activators can also be utilized. Inthis system, Parent 1 is transformed with an expression cassettecomprising an anther 5' regulatory region operably linked to thecauliflower mosiac virus translation activator (TAV). Parent 2 istransformed with an expression cassette comprising an anther specificpromoter operably linked to a dicistronic mRNA wherein the secondcistron encodes a cell toxin. Crossing parents 1 and 2 results in malesterile offspring as both cistrons of the dicistronic mRNA will betranslated in the presence of TAV. See, Bonneville et al., (1987), Cell,59:1135-1143; Futterer and Hohn, (1991), EMBO J., 10:3887-3896; Gowda etal., (1988), Proc. Natl. Acad, Sci., USA, 86:9203-9207; Scholthof etal., (1992), J. Virology, 66:3131-3139; and EP 298 918 filed Jul. 10,1987.

In some instances it may be useful to combine the use of more than onetransactivator in a single system. For example, transcriptionalactivation via T7 polymerase or GAL4/VP16 can be combined withtranslational activation. This combination may provide a tighter controlof unwanted expression of the toxin gene in the absence of thetransactivator.

Methodologies for the construction of plant expression cassettes as wellas the introduction of foreign DNA in to plants is generally describedin the art. Generally, for the introduction of foreign DNA into plantsTi plasmid vectors have been utilized for the delivery of foreign DNA aswell as direct DNA uptake, liposomes, electroporation, micro-injection,and the use of microprojectiles. Such methods had been published in theart. See, for example, Guerche et al., (1987) Plant Science 52:111-116;Neuhause et al., (1987) Theor. Appl. Genet. 75:30-36; Klein et al.,(1987) Nature 327:70-73; Howell et al., (1980) Science 208:1265; Horschet al., (1985) Science 227:1229-1231; DeBlock et al., (1989) PlantPhysiology 91:694-701; Methods for Plant Molecular Biology (Weissbachand Weissbach, eds.) Academic Press, Inc. (1988); and Methods in PlantMolecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc.(1989). It is understood that the method of transformation will dependupon the plant cell to be transformed.

It is further recognized that the components of the expression cassettemay be modified to increase expression. For example, truncatedsequences, nucleotide substitutions or other modifications may beemployed.

It may also be beneficial to remove nucleotides in the T7 promotersequence to prevent potential stem-loop structures in the RNA. Suchnucleotides can be removed, for example, using PCR technology as setforth below in the Experimental Section. Likewise, a poly A chain may beincluded in the expression cassette adjacent to the terminator. Forexample, in expression cassette 2, about 25 to about 90 A nucleotidesmay be inserted 5' to the T7 terminator.

Other methods such as transplicing may also be employed utilizing thesplice donor and splice acceptor sites of known genes such as the Adhlgene of maize. See. Dennis et al., (1984), Nucleic Acids Research,12:3983-4000. Such a system involves three expression cassettes. Thefollowing are typical cassettes which could be utilized in a T7 system.Cassette 1 comprises an anther-specific promoter operably linked to anucleotide sequence encoding a transactivator, e.g. T7 polymerase.

Cassette 2 comprises the target nucleotide sequence, T7 promoter,operably linked to a nucleotide sequence comprising a splice acceptorsite. The acceptor site is operably linked to a nucleotide sequencecomprising the 3' portion of the coding region of a polypeptide or RNAcapable of disrupting the formation of viable pollen.

Cassette 3 comprises a nucleotide sequence encoding a promoter capableof directing expression in anther tissue, operably linked to anucleotide sequence comprising the 5' coding region of the polypeptideor RNA capable of disrupting the formation of viable pollen which isoperably linked to a splice donor site. As discussed earlier, thecassettes may also comprise leader sequences, terminators, etc. Parentplant 1 can be stably transformed with cassette 1, or alternatively,cassettes 1 and 3 while Parent plant 2 will contain cassettes 2 and 3,or alternatively cassette 2, respectively. In either situation crossingParent 1 and Parent 2 results in male-sterile progeny.

Transformed plants are regenerated. The presence of the stablyintegrated expression cassette into the transformed parent plants may beascertained by southern hybridization techniques or PCR analysis, knownin the art. Expression of the transactivator may be determined byutilizing northern blot techniques.

Therefore, the present system can be utilized in any plant which can betransformed and regenerated. The method eliminates the necessity ofmanipulating floral structures and avoids the necessity of handemasculation and fertilization.

The parent plants containing the respective stably integrated expressioncassettes are both male fertile and can be made homozygous andmaintained indefinitely. To obtain male-sterile seed, homozygous linesof parent 1 and 2 are crossed using a technique such as detasseling ofone line and using the other as a pollinator, such that no self seed isproduced. The male-sterile offspring can then be utilized as femaleparents in any cross to produce hybrid seed. About 75% of the resultinghybrid seeds will give rise to male fertile plants. Thus, for thepurpose of producing hybrid seed, standard crossing of different lineswith the male-sterile plants and subsequent analysis of the progeny toselect a line with superior agronomic traits are performed. See,generally, International Patent Application Number WO 90/08828.

While the T7 polymerase system is useful in the above-described dualsystem for the production of male-sterile plants, it is also recognizedthat a T7 expression system can be utilized for high level expression ofnucleotide sequences in plants. The system also provides tissue-specificexpression or other selective expression of coding sequences in a plant.

In this manner, a single plant can be transformed with two expressioncassettes. A first expression cassette comprises a T7 polymeraseoperably linked to a promoter capable of directing expression in a plantcell. Any promoter capable of directing expression can be utilized andcan be chosen for specific expression; e.g. tissue-specific promoter,developmental stage-specific promoter, inducible promoter, etc. Specificpromoters, for example, include chemical inducible promoters (U.S.patent application Ser. No. 678,378) and seed specific promoters (Elliset al., (1988), Plant Mol. Biol., 10:203-214). As described earlier, thefirst expression cassette may additionally comprise nuclear locationsignals, terminator sequences, plant translational consensus sequences,etc.

The second expression cassette comprises a coding sequence operablylinked to a nucleotide sequence encoding T7 promoter. The secondexpression cassette may also comprise 5' leader sequences, terminatorsequences, etc.

When both expression cassettes have been stably integrated into a singleplant, the T7 polymerase will drive expression of the coding sequenceoperably linked to the T7 promoter.

It is recognized that the two expression cassettes may be part of asingle vector or nucleic acid sequence or may be housed on separatevectors. Likewise, while a single plant in most instances will betransformed with each cassette, it may be beneficial at times totransform one plant, parent 1, with expression cassette 1 and anotherplant, parent 2, with expression cassette 2 and obtain progeny with bothexpression cassettes by crossing parents 1 and 2.

Because transcription by T7 RNA polymerase is highly active, this systemmay be utilized to increase the production of specific gene productswhich are produced in low quantities in plants. The method is alsouseful for increasing tissue or other specific gene products. Generally,at least about a two fold to greater than a 100 fold, more specificallyabout 4 fold to about 50 fold, increase in expression can be seen usinga T7 system.

The T7 RNA polymerase is very selective for specific promoters that arerarely encountered in DNA unrelated to T7 DNA. Efficient terminationsignals are also rare. Therefore, the T7 RNA polymerase expressionsystem can make complete transcripts of almost any DNA that is placedunder control of a T7 promoter. Accordingly, the T7 expression systemcan be used to express a wide variety of products such as seed storageproteins with preferred amino acid composition; pharmaceutical proteins;proteins involved in starch, lipid or protein synthesis; insecticidal ordisease resistance proteins; proteins which increase the nutritionalquality of plants or seeds; antifungal, antibacterial or antiviralproteins; proteins that lead to the production of other proteins thatrender the plant resistant to insects or diseases; assembly proteins orproteins that are required for the production of other proteins; and thelike.

As discussed earlier, methods for manipulation of nucleic acid sequencesand for transformation and regeneration of plants are known in the art.

Having generally described the invention, the following examples areoffered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example 1--Addition of a plant translational consensus sequence to theT7 RNA polymerase gene

The translational start site of the T7 RNA polymerase gene (with theSV40 NLS) of pAR3283 (Dunn et al., Gene 68:259-255 (1988) was modifiedto include a plant translational consensus sequence (Joshi, C. P., NAR15, 6643-6653 (1987)). The BglII to NruI fragment of pAR3283 wasreplaced with a BglII--NruI PCR generated fragment in which the sequenceTAAACAATG, following the BglII site, replaced the sequence before the T7translational start site. The nucleotides after the translational startsite were not modified to conform to the plant consensus sequence(TAAACAATGGCT); SEQ ID NO: 1 because an asparagine to alaninesubstitution would result.

Example 2--Fusion of the 35S CaMV promoter to the T7 RNA polymerase gene

The T7 RNA polymerase gene containing the SV40 nuclear localizationsignal (NLS) and a plant translational consensus sequence was excised asa BglII--BamHI fragment and cloned into the BamHI site of pCIB710(Rothstein et al., Gene 53:153-161 (1987)). The resulting plasmid,pJS175, contains the 35S CaMV promoter, T7 RNA polymerase gene (SV40NLS, plant translational consensus sequence) and the 35S CaMV poly Aaddition site.

Example 3--T7 promoter/terminator constructions and fusions to the GUSgene

The T7 promoter and T7 terminator from pET-3 (Rosenberg et al., Gene 56,125 (1987)) was inserted as a BglII fragment into BamHI-cleaved pUC19 tomake pAT26 and into BamHI-cleaved bluescript SK to make pAT10. The 3'Sac I site of the GUS gene from pBI121 (Clontech) was adapted to containa Bam HI site and cloned into the Bam HI site between the T7 promoterand terminator of pAT10 to make pAT11.

In pAT27, the nucleotides +9 to +26 of the T7 promoter were removed byPCR from pAT26 in order to eliminate a potential stem-loop structure inthe RNA. A 35S CaMV poly A addition signal was inserted into the BamHIsite of pAT27 by adding a BglII site by PCR on the 3' end of thefragment, resulting in pAT28. The GUS gene from pAT11 was inserted intothe BamHI site of pAT28 to make pAT30 and into the BamHI site of pAT27to make pAT32. pJS261 was constructed by replacing the T7 terminator ofpAT26 with a BamHI--EcoRI fragment containing the 35S terminator, T7terminator from pAT28. A BamHI fragment containing the TEV leader-GUSgene from pAT31 was then inserted in the BamHI site.

Example 4--Translational constructs using the tobacco etch virus leader

The tobacco etch virus 5' nontranslated leader (nucleotides +6 to +143of the genomic RNA, Allison et al., Virology 154:9-20 (1986)), with aBamHI site on the 5' end and a NcoI site on the 3' end, wastranslationally fused to a GUS gene (NcoI--SacI fragment) intobluescript SK to make pAT29. The SacI site of pAT29 was adapted tocontain BamHI and the BamHI fragment containing the TEV leader-GUS genewas inserted into the BamHI site of pAT28 to make pAT31.

Example 5--Protoplast transformation and GUS fluorometric assays

Nicotiana tabacum protoplasts were transformed as described in Negrutiu,I. et al., PMB 8:363-373 (1987) and GUS fluorometric assays wereperformed as in Jefferson, R. A., PMB Reporter 5:387-405 (1987).

Methods for production of maize protoplasts are described in U.S. patentapplication Ser. No. 772,027 filed Oct. 4, 1991, herein incorporated byreference.

Example 6--Transcription from the T7 promoter in transient experiments

Maize protoplasts were cotransformed with the 35S promoter driving T7RNA polymerase (pJS175) and the T7 promoter/GUS gene/T7 terminator(pAT11). As a negative control, protoplasts were also cotransformed witha 35S promoter/luciferase gene (pCIB1700) and with pAT11. Protoplaststransformed with 35S/GUS (pCIB246) were a positive GUS control. RNA wasisolated from protoplasts according to the guanidiniumthiocyantate-phenol-chloroform method described by Goodall et al.,Methods in Enzymology 181:148-161 (1990). Duplicate northerns wereprobed with a T7 RNA polymerase and a GUS probe. Only RNA from theprotoplasts transformed with pJS175 and pAT11 hybridized to the T7 RNApolymerase probe. RNA from protoplasts transformed with pJS175 alone andwith pJS175/pAT11 hybridized to the GUS probe, showing that T7 RNApolymerase is transcribing off the T7 promoter in plant cells. GUS RNAlevels transcribed from the T7 promoter were 10-fold higher than thepCIB246 control.

Example 7--GUS expression using the Tobacco Etch Virus leader fortranslation of T7 transcripts

Tobacco protoplasts were cotransformed with the 35S CaMV promoterdriving T7 RNA polymerase (pJS175) and T7 promoter--GUS fusions with andwithout the TEV leader (pAT31, pJS261). GUS fluormotetric assays weredone (Table I). GUS enzyme activity (4-fold higher than the 35S/GUScontrol) was seen in T7 constructs only when the TEV leader was present.

                  TABLE I                                                         ______________________________________                                        Transient expression experiment using the TEV leader                          ______________________________________                                        pCIB 246                                                                             35S/GUS                                                                pJS 175                                                                              35S/T7 RNA polymerase                                                  pJS 179                                                                              355/luciferase                                                         pAT11  T7 promoter/GUS/T7 terminator                                          pJS261 T7 promoter/TEV leader/GUS/35S terminator/T7 terminator                pAT31  T7 promoter (stem loop removed)/TEV leader/GUS/35S                            terminator/T7 terminator                                               ______________________________________                                                   Specific GUS Activity                                                                         Fold increase over                                            (nm MU/μg protein/min.)                                                                    pCIB246                                            ______________________________________                                        pCIB 246   20.5 ± 4                                                        pJS 179/pAT11                                                                            0.021 ± 0.007                                                   pJS 175/pAT11                                                                            0.013 ± 0.004                                                   pJS 175/pJS261                                                                           14.17 ± 0.45 0.7                                                pJS 175/pAT31                                                                            80.8 ± 5     3.9                                                ______________________________________                                    

Example 8--Fusion of an anther-specific promoter to the T7 RNApolymerase gene

The T7 RNA polymerase gene containing the SV40 nuclear localizationsignal and a plant translational consensus sequence was excised as a BglII--Bam HI fragment and cloned into the Bam HI site of pLC250. InpLC250, a tapetal-specific tobacco anther promoter, ant32, was clonedinto the Sal I, Xba I sites of the Agrobacterium binary plasmid vectorpBI101 (Clontech, Palo Alto, Calif.). The GUS gene had previouslyremoved from pBI101 with Sma I, Sac I and the Sac I site had beenblunted. The resulting plasmid, pAT20, contains the ant32 tobacco antherpromoter, the T7 RNA polymerase gene (SV40 NLS, plant translationalconsensus sequence) and a nos terminator.

Example 9--Construction of plant transformation vectors containing ananther-specific promoter driving T7 RNA polymerase and the T7 promoterdriving the Diphtheria toxin gene

A plant transformation vector was constructed containing ananther-specific promoter driving T7 RNA polymerase and a T7 promoterdriving the Diphtheria toxin A-chain (DTA) coding sequence (Palmiter etal, Cell 50:435-443). A T7 promoter/TEV leader/DTA coding sequence/35Sterminator/T7 terminator cassette was made by excising the GUS gene frompAT30 with Bam HI and inserting in a TEV leader Bam HI--Nco I fragmentfrom pAT29 and a DTA coding sequence Nco I--Bgl II fragment, resultingin pTG28. pTG32 is a vector for plant transformation containing bothcomponents and was made by adaptoring the 3' Eco RI site of pTG28 toHind III and inserting the Hind III fragment into pAT20.

The anther-specific promoter driving T7 RNA polymerase and the T7promoter driving DTA can also be independently transformed into plantsand then crossed in order to produce male-sterile plants. pTG35 containsonly the T7 promoter driving DTA in a plant transformation vector andwas constructed by adapting the 3' Eco RI site of pAT28 to Sal I andcloning into the Sal I site of the plant transformation vector pCIB905.Plants transformed with pTG35 can be crossed to pAT20 transformants.

Example 10--Production of transgenic plants

Tobacco leaf discs were transformed with pTG32, pAT20 and pTG35 asdescribed in Horsch et al., Science 227:1229-1231 (1985) and thepresence of transforming DNA was confirmed using PCR.

Example 11--Analysis of plants transformed with an anther-specificpromoter driving T7 RNA polymerase and the T7 promoter driving DTA

The flower morphology of 13 plants transformed with pTG32 was observed.11 plants were male-sterile and 9 of the 11 plants were shown to befemale-fertile by backcrossing with wild-type tobacco.

Example 12--Plant transformation vectors for GUS expression from the T7promoter

The 35S CaMV promoter driving the T7 RNA polymerase and the T7 promoterdriving the GUS gene were cloned into a plant transformation vector. Asa control, the 35S CaMV promoter driving luciferase and the T7 promoterdriving the GUS gene were also cloned. The T7 promoter (stem loopremoved)/TEV leader/GUS gene/35S terminator/T7 terminator were removedfrom pAT31 with Xba I, Eco RI and cloned into the plant transformationvector pCIB200 in pAT34. The 5' Sac I site of the 35S promoter/T7 RNApolymerase/nos terminator fragment was adapted to contain Xba I (Sac Isite destroyed) and cloned into the Xba I site pAT34 to make pAT35. Forthe control, a luciferase gene was cloned into the BamHI site of pCIB770(Rothstein et al., Gene 53:153-161 (1987)) in pAT36. In pAT37, the EcoRIsite of pAT31 was adapted to Sal I (Eco RI destroyed) and the Sal Ifragment containing the T7 promoter/TEV leader/GUS gene/35Sterminator/T7 terminator was cloned into the Sal I site of pAT36. Inboth pAT35 and pAT37, clones were chosen which have transcription of thetwo genes in opposite orientations away from each other.

Example 13--Anti-sense inhibition using T7 polymerase and T7 promoters

In pCIB3217, the T7 promoter was inserted in an anti-sense directionafter a 35S promoter/GUS/35S terminator cassette in puc 19. Thiscassette is cloned into a plant transformation vector and is crossed toa plant transformed with 35S promoter/T7 RNA polymerase (pCIB3210). GUSenzyme activity of progeny carrying both T7 polymerase and the GUSgene/anti-sense T7 promoter is compared to progeny carrying only the GUSgene/anti-sense T7 promoter.

Example 14--Construction of vectors containing the GAL4 bindingsite/minimal 35S CAMV promoter fused to GUS and Diphtheria toxin

The GAL4 consensus binding site (Giniger et al., Cell 40:767-774 (1985)was fused to the CAMV 35S minimal promoter (-46 to +1, Benfey et al.,EMBO 9:1677-1684 (1990)) by incorporating the binding site into a PCRprimer. The PCR generated band containing the binding site and theminimal promoter contained HindIII, XbaI ends and was cloned intopBI101. pLP3 contains the GAL4 binding site/minimal 35S promoter/GUSgene/nos termininator in a plant transformation vector. This cassettewas excised from pLP3 with HindIII, EcoRI and cloned into bluescript tomake pLP4.

The GAL4 binding site/minimal 35S promoter was fused to the DTA codingsequence. The GUS gene was first removed from pBI101 by excising withSmaI and SacI, blunting the SacI site, and religating the plasmid backtogether. The GAL4 binding site/minimal 35S promoter was cloned into theHindIII, XbaI sites and the DTA gene was cloned as a BglII fragment intothe BamHI site of the vector. pLP1 contains the GAL4 binding site/35Sminimal promoter/DTA coding sequence/nos terminator in a planttransformation vector.

Example 15--GUS expression using the GAL4/VP16 transactivator

Maize protoplasts were cotransformed with a 35S promoter/GAL4/VP16 gene(pGAL4/VP1--Goff et al., (1991). Genes and Development, 5:298-309) and aGAL4 binding site/minimal 35S promoter/GUS gene (pLP4). GUS fluorometricassays were performed (Table II). GUS enzyme activity was 20 fold higherfrom the GAL4 binding site/minimal 35S promoter when the GAL4/VP16transactivator was present.

                  TABLE II                                                        ______________________________________                                        Transient expression experiment using GAL4/VP16                               transactivation                                                               ______________________________________                                        pLP4       GAL4 binding site/minimal 35S promoter/GUS                         pGAL4/VP1  35S promoter/Adhl intron/GAL4/VP16                                 ______________________________________                                                      Specific GUS Activity                                                                       Fold increase                                                   (nm MU/μg protein)                                                                       over pLP4                                         ______________________________________                                        no DNA        0.02 ± 0.0                                                   pLP4          0.26 ± 0.15                                                  pLP4/pGAL4/VP1                                                                              5.25 ± 1.20                                                                              20                                                ______________________________________                                    

Example 16--Fusion of an anther promoter to GAL4/VP16

The GAL4/VP16 fusion was excised as a BamHI fragment from pGAL4/VP1 andinserted into the BamHI site of pLC250. The resulting plasmid, pLP2,contains an anther-specific promoter/GAL4/VP16/nos terminator in a planttransformation vector. Transformants containing pLP2 can be crossed toplants transformed with pLP3 in order to get activation of the GUS geneor to plants transformed with pLP1 in order to produce male-sterileplants.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "plant consensus sequence                            from example 1"                                                               (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TAAACAATGGCT12                                                                __________________________________________________________________________

What is claimed is:
 1. A vector comprising an expression cassettecomprising an anther-specific 5' regulatory region operably linked to anucleotide sequence encoding a transactivator polypeptide not naturallyassociated with plants, wherein said transactivator polypeptide turns ontranscription that is otherwise off in the absence of saidtransactivator polypeptide.
 2. A vector comprising an expressioncassette comprising a target nucleic acid sequence operably linked to anucleotide sequence which encodes anti-sense RNA which disrupts theformation of viable pollen or a polypeptide which disrupts the formationof viable pollen, wherein said target nucleic acid sequence is capableof being activated by a transactivator polypeptide not naturallyassociated with plants, and wherein said transactivator polypeptideturns on transcription that is otherwise off in the absence of saidtransactivator polypeptide.